This application claims priority from Australian Provisional Application Ser. No. PQ 6530 filed Mar. 28, 2000.
1. Technical Field
This invention relates to incubators and more particularly incubators for cell culturing, in particular culturing embryos of mammalian species.
2. Background of the Invention
Some cells growing in a liquid medium produce exogenous and growth factors which surround the cell in the liquid medium. In the growing of such cells in vitro it is important not to immediately flush these exogenous and growth factors when changing the medium around the cell.
It is the object of this invention to provide an incubator well for the growing of cells such as embryos which have favorable growing conditions.
The invention is said to reside in a perfusion incubator, including a fluid supply, a fluid conditioning unit, at least one well assembly and a well assembly heating unit, a pump such as a peristaltic pump, and preferably a fluid collection unit, the well assembly including at least one well, each well having a fluid inlet and a fluid outlet, each fluid inlet being positioned at a point in the well above the cell and each fluid outlet being positioned above its respective fluid inlet above the cell (such as an embryo) to be cultured in a lower portion of the well, the fluid inlet connected to the fluid supply and the fluid outlet connected to the fluid collection unit via the peristaltic pump whereby on the placing of the specimen cell to be cultured in each well and flowing fluid through the well, culturing of the cell can occur.
There may be further included an illumination device so that the cell being cultured in the well assembly can be observed by means of a microscope. There may be further included a microscope mount associate with the perfusion incubator.
Each well may include means to provide a flow path from the fluid inlet to the fluid outlet within the well so that fluid flow is not directly around the cell in the well.
Preferably, each fluid inlet is positioned so as to allow a tangential entry of fluid to the well at a mid point in the well, and the fluid outlet being positioned above the fluid inlet with the cell to be cultured in a lower portion of the well, whereby flow of fluid in the well is formed by this construction into a vortex which will tend to draw fluid from around the cell without direct flow over the cell.
Each well can have a stepped side wall defining an upper chamber and a smaller lower chamber with a lid which in use is adapted to extend partially into the upper chamber.
Each lid may be made of a substantially transparent material so as to allow for viewing of the cell in the lower chamber.
The well assembly may be transparent so that the cell can be illuminated from below.
The peristaltic pump can provide a flow rate of fluid through each well of between 40 microliters per hour up to 4000 microliters per hour when in flush mode.
The fluid conditioning unit may be operated at a temperature of approximately 0.5xc2x0 C. above the operating temperature of the well assembly heating unit whereby increase in solubility of gases in the fluid because of the 0.5xc2x0 C. temperature drop upon entry into a well, and decrease in solubility because of reduced pressure in the well due to suction of the peristaltic pump, are both compensated for and gas bubbles do not form in the culture well.
In an alternate form the invention is said to reside in a perfusion incubator well assembly having a body, at least one well in the body, the or each well having a stepped side well defining an upper chamber and a smaller lower chamber and a lid, a fluid inlet to the or each well and fluid outlet from the or each well, the fluid inlet being positioned so as to allow tangential entry of fluid to the well at a lower portion of the upper chamber and the fluid outlet being positioned above the fluid inlet.
A well closure such as a lid may be adapted to extend partially into the upper chamber, and may include an O-ring seal and be made of a substantially transparent material so as to allow viewing of an cell in the lower chamber.
The body may be made from a substantially transparent material so that with illumination from below, the cell being cultured can be viewed.
The fluid inlet and the fluid outlet to the or each well may be formed by apertures formed in the body.
It can be seen, therefore, that by this invention the perfusion incubator is a system which based on the principle of perfusion of specifically designed culture fluid, provides a suitable environment for the production, development and storage of pre-implantation embryos from mammalian species. The system maintains purpose-built culture wells at a pre-set temperature while perfusing carbon dioxide enriched culture media over the cells.